PROJECT SUMMARY Mitochondria are endosymbiotic organelles that possess a residual genome (mtDNA) encoding a handful of proteins and ribosomal and transfer RNAs essential for their functions. Human cells possess 100-1000s of mtDNAs, actively condensed into nucleoids - protein-DNA structures that are the cellular unit of mtDNA inheritance ? distributed within dynamic mitochondria ?syncytia?. Although the molecular players involved in mtDNA replication and packaging have been described, much less is understood about how at the cellular level nucleoids are distributed within mammalian cells to meet the needs for mitochondrial function, for example, how they are selected for mtDNA replication and how the cellular copy number of mtDNA is controlled. We discovered that in human cells, nucleoids engaged in mtDNA replication are spatially linked to a small subset of ER-mitochondria contact sites destined for mitochondrial division and motility. We found that the successive events of mtDNA replication, mitochondrial division and mitochondrial motility function together in a pathway that preferentially distributes nascent mtDNA in cells, which we term ER-linked mtDNA transmission. In this grant, we explore the underlying mechanisms of this ER-linked mtDNA transmission pathway by addressing the cell biology and behavior of the mammalian nucleoid. New information in this understudied area of cell biology will more accurately reveal the etiology of human metabolic diseases caused by mutations in mtDNA and in nuclear genes that affect mtDNA maintenance and in aging and neurodegenerative disorders, which are also linked to defective mtDNA maintenance and mitochondrial and ER dysfunction.